Part of the research and development cycle of pharmaceuticals is determining how an experimental drug interacts with the body. This is a critical step in learning not only if the pharmaceutical is effective for its intended purpose but also what other unintended side effects it may cause. One way for identifying the effects of a new pharmaceutical is to conduct laboratory experiments that expose the pharmaceutical to biological samples (e.g., proteins) and then observing the results of that interaction. Among other things that may be observed during these experiments, the shape of the sample may change and these changes in shape can provide an indication of the effects that the pharmaceutical would have on a body. In other words, these structural characterization studies show the site and strength of drug molecule binding. To ensure that the results of these tests are accurate, it is important to begin the experiment with a biological sample that is as close to its natural state and shape as possible with high structural and functional integrity. If the shape of a biological sample is altered or damaged before a test is conducted, the results of the test may not accurately reflect how the pharmaceutical being tested would affect the body in real life. Similar arguments are also valid in other fields such as materials science that involve looking at component interactions in the liquid state.
Cryogenic electron microscopy (cryo-EM) is an electron microscopy technique involving the imaging of biological materials in a transmission electron microscope under cryogenic conditions. In cryo-EM, high-voltage electrons are generated in a vacuum by an electron source (i.e., an electron gun). Those electrons are focused into a fine beam and are then directed towards and through a sample located on a movable stage. After passing through the sample, the electrons either scatter or hit an image recording system that includes an electron detector to generate an image. However, before any imaging or analysis can occur in the cryo-EM process, the samples must be prepared. During the sample preparation stage, sample proteins in an aqueous environment are captured in a thin layer of vitreous ice by being cooled very quickly (generally, in less than a millisecond) to cryogenic temperatures. When samples are prepared properly, the vitreous ice layer can trap biological matter in its natural form and provides a thin (generally, less than 3 micrometers thick), clear sample that is well suited for cryo-EM imaging and analysis. Cryo-EM may also be used in other scientific fields, including materials science, nanomedicine, and renewable energy.
Thus, sample preparation is a very important step in cryo-EM analyses. However, sample preparation is often complex, difficult, and costly. One common issue is the inability to reliably and precisely control the thickness of the vitreous ice formed when preparing a sample on a cryo-EM grid. Since electrons must transmit through an EM sample for an image to be formed, it is necessary that the sample be thin enough to transmit sufficient electrons to form an image with minimum energy loss and a high enough signal-to-noise ratio. On the other hand, if the sample is too thin, the sample may not be fully encapsulated by the vitreous ice layer and may extend through and become exposed at the water-air interface, which can cause their shape or composition to be adversely impacted. Proteins can aggregate and become grouped too closely together if the ice layer is too thin, or they may become disassociated (i.e., torn apart) or spread too far apart from or stack on top of one another if the ice layer is too thick. Other issues, such as the formation of ice artifacts and crystallization within the ice that cloud the ice, can make obtaining an image difficult or impossible. Therefore, carefully forming the vitreous ice layer with a particular thickness and clarity is critical to obtaining good samples that are suitable for use in cryo-EM imaging and analysis.
Conventional vitrification processes rely on trial and error to achieve an acceptable sample. Typically, several cryo-EM samples are prepared on EM grids under a variety of conditions, with the hope that one of those conditions will produce a vitrified sample having the desired ice thickness and clarity. With reference to FIGS. 1-3, there is illustrated an example of a conventional EM grid 100 that may be used to suspend biological samples. Grid 100 includes a flat disk 102, which is provided with an array of grid openings 104 that are formed by intersecting metallic rails 106. These rails 106 are made from a material having high thermal conductivity such as copper, nickel, aluminum, etc. A single grid opening 104′ is highlighted in FIG. 1 and is enlarged in FIG. 2. A film 108, sometimes called a holey carbon film, is placed on top of and is adhered to the disk 102 and covers the grid openings 104. The film 108 is provided with an array of very small holes 110 that extend through the film and across the entire surface of the disc 102.
These grids 100 are commonly used in a conventional sample preparation method known as the blotting and plunge freezing method, which can be done manually or semi-automatically with devices currently on the market. In preparing a sample for cryo-EM imaging and analysis, a droplet of a sample material is often deposited onto the film by hand using a pipette. A cross section of two of the holes 110 of grid opening 104′ is shown in FIG. 3. As seen there, the sample solution 112 fills the holes 110 but a large amount of the solution collects on top of the film 108. At the blotting step, filter paper is brought into contact with the sample solution 112 and a portion of the sample solution is absorbed into the filter paper. The left hole 110 shown in FIG. 3 shows the sample solution 112 before the filter paper is used. The right hole 110 shown in FIG. 3 shows the sample solution 112 after a portion of the sample solution 112 has been removed from the grid 102 at the blotting step. The grid 100 is then vitrified by plunge freezing into a cryogen, such as liquid ethane, liquid propane, or a mixture of the two cooled by liquid nitrogen.
FIGS. 4-6 are enlarged side views of the right hole 110 shown in FIG. 3 under three different scenarios. In each case, sample solution 112 is held in the hole 110 by surface tension (i.e., capillary action) and a meniscus having a peak 114 formed at the center of the layer of sample solution on both the top and bottom of the layer of sample solution. The vertical distance between these peaks 114 defines a height H of the ice layer. In FIG. 4, too much sample solution 112 was left remaining at the blotting stage, which resulted in a vitreous ice layer having a thickness H that is too great. This is evidenced by the stacking of proteins 116 on the left-hand side and the dissociated protein shown on the right-hand side. In FIG. 5, an insufficient amount of sample solution 112 was left at the blotting stage, which resulted in an ice layer having a thickness H that is too small. This is evidenced by the exposure of the proteins 116, which extend through the water-air interface 118. In FIG. 6, an ideal amount of sample solution 112 was left at the blotting stage, which resulted in an ice layer having an ideal thickness H. This is evidenced by proteins 116 that are fully encapsulated by the ice layer and are well-dispersed throughout. Ideally, proteins 116 are homogeneous and well-dispersed in a single layer across throughout the vitreous ice layer and adopt random orientations. These random orientations allow the proteins to be viewed from multiple angles in a single view, enabling the three-dimensional structural reconstruction at a later step.
As shown above, the conventional blotting and plunge freezing method is unreliable, labor intensive, and slow. Each stage of sample preparation, namely pipetting, blotting, and plunge freezing, is carried out sequentially or by hand. The actual amount of time separating each of these steps may be only seconds, but it is long enough for the samples to be adversely impacted as molecules tumble around in solution. For example, when sample solutions are initially deposited onto a grid, they may have well-dispersed and randomly-oriented individual proteins. However, while the blotting occurs and before the plunge freezing step, those proteins may coalesce, disperse, readjust their configurations in solution, and adopt a preferential alignment (i.e., proteins align themselves in a particular manner and are not randomly oriented). Each of these behaviors negatively impacts the sample and makes EM imaging and the determination of protein structures more difficult.
Another major drawback to this conventional process is the cost associated with waste sample material. Generally, a 2-4 μl sample volume is required to prepare a single sample grid 100. However, 99.9% of the sample volume is lost during grid preparation. Much of this loss occurs at the blotting stage of sample preparation, but evaporation is also a cause of loss of the sample solution. Sample solutions are often difficult and expensive to obtain due to extensive work in synthesis, extraction, and purification, etc. For that reason, attempts have been made to reduce these losses. For example, samples are sometimes prepared in an environment having a high humidity level, such as in an enclosed chamber, such that sample loss due to evaporation is minimized or eliminated. However, as explained above, EM imaging occurs in a vacuum. For that reason, it has been impossible to prepare a sample using the conventional blotting and plunge freezing method and then image that sample in the same environment.
Therefore, what is needed, is an improved method and apparatus for preparing biological samples for cryo-EM imaging and analysis.